Drugs of abuse, including cocaine, amphetamine (AMPH), and heroin, elevate extracellular dopamine (DA) levels in the brain, thereby altering the activity͞plasticity of reward circuits and precipitating addiction. The physiological release of DA occurs through the calcium-dependent fusion of a synaptic vesicle with the plasma membrane. Extracellular DA is cleared by uptake through the Na ؉ ͞Cl ؊ -dependent DA transporter (DAT). In contrast, the substrate AMPH induces nonvesicular release of DA mediated by DAT. Extracellular AMPH is generally believed to trigger DA efflux through DAT by facilitating exchange for cytosolic DA. Here, in outside-out patches from heterologous cells stably expressing DAT or from dopaminergic neurons, by using ionic conditions in the patch pipette that mimic those produced by AMPH stimulation, we report that AMPH causes DAT-mediated DA efflux by two independent mechanisms: (i) a slow process consistent with an exchange mechanism and (ii) a process that results in rapid (millisecond) bursts of DA efflux through a channel-like mode of DAT. Because channel-like release of DA induced by AMPH is rapid and contains a large number of DA molecules, with a single burst of DA on par with a quantum of DA from exocytotic release of a vesicle, this burst mode of release may play a role in the synaptic actions and psychostimulant properties of AMPH and related compounds. Unlike AMPH, the endogenous substrate DA, when present on both sides of the plasma membrane, inhibits this channel-like activity, thereby suggesting that the DAT channel-like mode cannot accumulate DA against a concentration gradient.patch clamp ͉ amperometry D opamine (DA) transporter (DAT) is a member of a gene family that includes norepinephrine transporter (NET), serotonin transporter (SERT), and ␥-aminobutyric acid transporter 1 (GAT-1) (1-3). These neurotransmitter transporters are thought to play an important role in the reuptake (3, 4) and release (5, 6) of neurotransmitters. Their mechanism of transport is often described by using an alternating access model (7). In this model, the binding of cargo (DA, Na ϩ , and Cl Ϫ ) to an extracellularly oriented transporter induces a conformational rearrangement to an intracellularly oriented transporter from which the cargo is released into the cytosol, thereby completing the transport process. Consistent with this model, the recent crystal structure of the nonhomologous secondary transporter lactose permease (8) showed the transporter in what was inferred to be the ''inward-facing state'' with substrate bound within a cytoplasmic vestibule and no access route to the ''extracellular'' space.In the case of DAT, DA transport generates an electrical current because of the net movement of positive charge into the cell (9). DA efflux induced by the psychostimulant amphetamine (AMPH) is believed to result from the ability of AMPH to reverse this inward transport process, in that the inward transport of AMPH by DAT increases the number of ''inward-facing'' transporter binding sites and there...

The soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein syntaxin 1A (SYN1A) interacts with and regulates the function of transmembrane proteins, including ion channels and neurotransmitter transporters. Here, we define the first 33 amino acids of the N terminus of the dopamine (DA) transporter (DAT) as the site of direct interaction with SYN1A. Amphetamine (AMPH) increases the association of SYN1A with human DAT (hDAT) in a heterologous expression system (hDAT cells) and with native DAT in murine striatal synaptosomes. Immunoprecipitation of DAT from the biotinylated fraction shows that the AMPH-induced increase in DAT/SYN1A association occurs at the plasma membrane. In a superfusion assay of DA efflux, cells overexpressing SYN1A exhibited significantly greater AMPH-induced DA release with respect to control cells.By combining the patch-clamp technique with amperometry, we measured DA release under voltage clamp. At Ϫ60 mV, a physiological resting potential, AMPH did not induce DA efflux in hDAT cells and DA neurons. In contrast, perfusion of exogenous SYN1A (3 M) into the cell with the whole-cell pipette enabled AMPH-induced DA efflux at Ϫ60 mV in both hDAT cells and DA neurons. It has been shown recently that Ca 2ϩ /calmodulin-dependent protein kinase II (CaMKII) is activated by AMPH and regulates AMPH-induced DA efflux. Here, we show that AMPH-induced association between DAT and SYN1A requires CaMKII activity and that inhibition of CaMKII blocks the ability of exogenous SYN1A to promote DA efflux. These data suggest that AMPH activation of CaMKII supports DAT/SYN1A association, resulting in a mode of DAT capable of DA efflux.

Highlights Narsoplimab down-modulates SARS-CoV-2-induced activation of the lectin pathway and endothelial cell damage. Narsoplimab can reduce the thrombotic risk of Covid-19 patients. All patients treated with narsoplimab improved and survived without any drug-related adverse events.

Bergamo province was badly hit by the coronavirus disease 2019 (COVID-19) epidemic. We organised a public-funded, multidisciplinary follow-up programme for COVID-19 patients discharged from the emergency department or from the inpatient wards of ‘Papa Giovanni XXIII’ Hospital, the largest public hospital in the area. As of 31 July, the first 767 patients had completed the first post-discharge multidisciplinary assessment. Patients entered our programme at a median time of 81 days after discharge. Among them, 51.4% still complained of symptoms, most commonly fatigue and exertional dyspnoea, and 30.5% were still experiencing post-traumatic psychological consequences. Impaired lung diffusion was found in 19%. Seventeen per cent had D-dimer values two times above the threshold for diagnosis of pulmonary embolism (two unexpected and clinically silent pulmonary thrombosis were discovered by investigating striking D-dimer elevation). Survivors of COVID-19 exhibit a complex array of symptoms, whose common underlying pathology, if any, has still to be elucidated: a multidisciplinary approach is fundamental, to address the different problems and to look for effective solutions.

The human dopamine transporter (hDAT) regulates synaptic dopamine (DA) levels and is the site of action of abused and therapeutic drugs. Here we study the effect of a threonine residue (Thr62 in hDAT) that is highly conserved within a canonical phosphorylation site (RETW) in the juxtamembrane Nterminal region of monoamine transporters. In stably transfected human embryonic kidney 293T cells, expression of T62D-hDAT was reduced compared with hDAT or T62A-hDAT. T62D-hDAT displayed dramatically reduced [ 3 H]dopamine uptake but exhibited a higher basal dopamine efflux compared with hDAT or T62A-hDAT, as determined by measurements of can rescue reduced DA uptake in mutant transporters that are predominantly inward-facing, micromolar concentrations of Zn 2ϩ markedly potentiated [ 3 H]dopamine uptake in T62D-hDAT and permitted the measurement of amphetamine-stimulated dopamine efflux. These results suggest that T62D-hDAT prefers an inward-facing conformation in the transition between inward-and outward-facing conformations. For T62A-hDAT, however, the measured 50% reduction in both [ 3 H]dopamine uptake and [ 3 H]dopamine efflux was consistent with a slowed transition between inward-and outward-facing conformations. The mechanism underlying the important functional role of Thr62 in hDAT activity suggested by these findings is examined in a structural context using dynamic simulations of a threedimensional molecular model of DAT.The strength and duration of dopaminergic neurotransmission is tightly controlled by the dopamine transporter (DAT), which mediates reuptake of synaptic dopamine (DA) (Chen and Reith, 2000) and is a target for therapeutic drugs and abused psychostimulants (Sulzer et al., 2005). Amphetamine (AMPH), a substrate for DAT, competitively inhibits DA reuptake and elicits outward transport of DA by reversal of the transporter (Sulzer et al., 2005), purportedly by an exchange diffusion mechanism (Fischer and Cho, 1979;Sulzer et al., 2005). This model assumes that the transporter transitions between two primary conformational states-an "outwardfacing" conformation favoring substrate binding and influx, and an "inward-facing" conformation in which the binding sites are available to the intracellular milieu and which favors substrate efflux. In the absence of substrate, the transporter favors an outward-facing conformation ready to bind

This consensus on core elements for hospital AMS programmes is relevant to both high- and low-to-middle-income countries and could facilitate the development of national AMS stewardship guidelines and adoption by healthcare settings worldwide.

Precise patterns of connectivity are established by different types of afferents on a given target neuron, leading to well-defined and non-overlapping synaptic territories. What regulates the specific characteristics of each type of synapse, in terms of number, morphology, and subcellular localization, remains to be understood. Here, we show that the signaling pathway formed by the secreted complement C1Q-related protein C1QL1 and its receptor, the adhesion-GPCR brain angiogenesis inhibitor 3 (BAI3), controls the stereotyped pattern of connectivity established by excitatory afferents on cerebellar Purkinje cells. The BAI3 receptor modulates synaptogenesis of both parallel fiber and climbing fiber afferents. The restricted and timely expression of its ligand C1QL1 in inferior olivary neurons ensures the establishment of the proper synaptic territory for climbing fibers. Given the broad expression of C1QL and BAI proteins in the developing mouse brain, our study reveals a general mechanism contributing to the formation of a functional brain.

Heterozygous and rare homozygous mutations in PRoline-Rich Transmembrane protein 2 (PRRT2) underlie a group of paroxysmal disorders including epilepsy, kinesigenic dyskinesia episodic ataxia and migraine. Most of the mutations lead to impaired PRRT2 expression and/or function. Recently, an important role for PRTT2 in the neurotransmitter release machinery, brain development and synapse formation has been uncovered. In this work, we have characterized the phenotype of a mouse in which the PRRT2 gene has been constitutively inactivated (PRRT2 KO). β-galactosidase staining allowed to map the regional expression of PRRT2 that was more intense in the cerebellum, hindbrain and spinal cord, while it was localized to restricted areas in the forebrain. PRRT2 KO mice are normal at birth, but display paroxysmal movements at the onset of locomotion that persist in the adulthood. In addition, adult PRRT2 KO mice present abnormal motor behaviors characterized by wild running and jumping in response to audiogenic stimuli that are ineffective in wild type mice and an increased sensitivity to the convulsive effects of pentylentetrazol. Patch-clamp electrophysiology in hippocampal and cerebellar slices revealed specific effects in the cerebellum, where PRRT2 is highly expressed, consisting in a higher excitatory strength at parallel fiber-Purkinje cell synapses during high frequency stimulation. The results show that the PRRT2 KO mouse reproduces the motor paroxysms present in the human PRRT2-linked pathology and can be proposed as an experimental model for the study of the pathogenesis of the disease as well as for testing personalized therapeutic approaches.